Long wavelength long lifetime luminophores

ABSTRACT

A new approach is described to making luminophores which display long emission wavelengths, long decay times, and high quantum yields. These luminophores are covalently linked or otherwise closely associated pairs with a long lifetime resonance energy transfer (RET) donor e.g., a ruthenium (Ru) metal-ligand complex, and a long wavelength acceptor, e.g., Texas Red. The donor and acceptor can be covalently linked by, e.g., poly-proline spacers. The long lifetime donor results in a long lived component in the acceptor decay which is due to RET. The quantum yield of the luminophores approaches that of the higher quantum yield acceptor, rather than the lower quantum yield typical of metal-ligand complexes. The emission maxima and decay time of such tandem luminophores can be readily adjusted by selection of the donor, acceptor and distance between them. Luminophores with these useful spectral properties can also be donor-acceptor pairs brought into close proximity by some biochemical association reaction. Luminophores with long wavelength emission and long lifetimes have numerous applications in biophysics, clinical diagnostics, DNA analysis and drug discovery.

[0001] This work was supported by NIH grant NCRR-08119 and GM 35154; thegovernment may have rights in this invention.

BACKGROUND OF THE INVENTION

[0002] In fluorescence spectroscopy the information available from anexperiment is related to the spectral properties of the fluorophore. Forexample, the anisotropy decay of fluorophores which display nanosecond(ns) decay times can be used to measure motions on the ns timescale. Agood number of fluorophores have become available which display red ornear infrared (NIR) emission [1-2]. Such probes are widely used in thebiochemical and medical applications of fluorescence, including proteinlabeling, chromatography, measurements in blood, noninvasive medicaltesting, DNA sequencing and analysis and in vivo measurements [3-13].Many of the red/NIR fluorophores display high extinction coefficientsand good quantum yields, both of which indicate the absorportion andemisson electronic transitions are strongly-allowed. Consequently, thedecay times of the red/NIR probes are typically below 4 ns and oftenbelow 1 ns, as is predicted by theory [14]. These fluorophores typicallydisplay small Stokes' shifts, and scattered light is most difficult toeliminate at wavelengths close to the excitation wavelength.

[0003] If slower motions on the μs timescale are of interest then it isnecessary to use fluorophores which display μs decay times. Furthermore,intracellular fluorophores which require UV excitation result in abackground of undesired emission due to the intrinsic fluorescence ofcells and tissues. This autofluorescence from biological samples ismostly on the ns timescale and its intensity decreases at longerexcitation and emission wavelengths. The signal-to-background ratiocannot be significantly improved by gated detection after the excitationpulse. Hence, the signals detected with red or NIR probes can beaffected by scattered light and/or sample autofluorescence.

[0004] For these reasons, for example, there is a need for infraredfluorophores which display long excitation and long emission wavelengthsand long decay times and preferably high quantum yields.

SUMMARY OF THE INVENTION

[0005] This invention relates to red/NIR luminophores which display bothlong decay times and high quantum yields and preferably large Stokesshifts.

[0006] In one aspect, this invention provides a method of providing aprobe which emits luminophore radiation in the range of a wavelength λ₁of about 400 nm to about 1200 nm with a high quantum yield Q₁ and a halflife greater than about 25 ns, comprising placing a donor molecule D,which per se emits radiation of a wavelength less than λ₁ with a quantumyield substantially lower than Q₁, in close association with an acceptormolecule A sufficient for resonant energy transfer from D to A, as aresult of which D resonantly transfers energy to A and A emits saidluminophore radiation.

[0007] In another aspect this invention provides a luminophorecomprising a donor portion (D) in close association with an acceptorportion (A) sufficient for resonant energy transfer from D to A, whereinupon excitation by external electromagnetic radiation of a wavelengthshorter than λ₁, said luminophore emits luminophore radiation of awavelength longer than λ₁, which is in the range of about 400 to about1200 nm with an emission lifetime τ₁ and a quantum yield Q₁,

[0008] wherein when D is not in said close association with A, itabsorbs radiation of a wavelength λ₂ shorter than λ₁ and thereafteremits radiation with a quantum yield Q₂ less than about 0.2,

[0009] wherein when said donor portion D is in said close associationwith A and is excited by electromagnetic radiation of wavelength shorterthan λ₁, it resonantly transfers energy to said acceptor portion A whichthen resonantly emits said luminophore radiation, and wherein saidquantum yield Q₁ is substantially greater than Q₂.

[0010] For example, this invention provides a compound of the formula

D-L-A

[0011] wherein D is a donor metal ligand complex having a quantum yieldless than about 0.2 for emission in the wavelength range of greater thanabout 400 nm;

[0012] A is an acceptor of energy resonantly transferred from D which isthen emitted in the wavelength range of about 400 to about 1200 nm; andL is a spacer of a length effective for resonant energy transfer betweenD and A.

[0013] In another aspect, this invention provides a chemical compoundmarked with a covalently bonded detectable label which is a compoundabove and provides the corresponding methods of labeling compounds andidentifying the latter in a mixture of compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Various other features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanyingdrawings, in which like reference characters designate the same orsimilar parts throughout the several views, and wherein:

[0015]FIG. 1 shows the chemical structure of a Ru MLC covalently linkedto Texas Red (D-A); wherein the donor-alone control has the sulfhydrylgroup blocked with iodoacetamide and the acceptor alone was the peptidewithout the MLC group.

[0016]FIG. 2 shows a Jablonski diagram for an irreversible excited stateprocess.

[0017]FIG. 3 shows the effect of energy transfer efficiency on the totalquantum yield.

[0018]FIG. 4 shows the simulated time-dependent decays of the donor andacceptor, each alone and in a D-A pair; for these simulations _(τ) _(D)⁰=1000 ns and _(τ) _(A) ⁰=10 ns.

[0019]FIG. 5 shows the emission spectra of the Ru-(pro)₆ donor (D) theTR acceptor (A) and the covalently linked pair (D-A) in aqueous buffer.

[0020]FIG. 6 shows the absorption (top) and excitation spectra (bottom)of Ru-pro)₆(D), TR(A), and Ru-(pro)₆-TR(D-A) in aqueous buffer.

[0021]FIG. 7 shows the ratio of the absorption spectra (top) andemission spectra (bottom) of the D-A pair divided by that of theacceptor in aqueous buffer.

[0022]FIG. 8 shows the frequency domain intensity decays of the donoralone (D), acceptor alone (A) and of the covalently linked D-pro₆-A pairin aqueous buffer (top) and in propylene glycol (bottom).

[0023]FIG. 9 shows the reconstructed time-domain intensity decays of thedonor alone (D), acceptor alone (A) and the covalently linked pair (D-A)in water (top) and in propylene glycol (bottom); the solid line t_(DA)is for D-pro₆-A and the dashed-dotted (-•-•-) line t_(DA) is forD-pro₈-A.

[0024]FIG. 10 shows the frequency domain intensity decays of the donoralone (D), acceptor alone (A) and of the covalently linked D-pro₈-cys-Apair in the aqueous buffer (top) and in propylene glycol (bottom).

[0025]FIG. 11. A potential long wavelength, long lifetime luminophorebased on a long lifetime donor (D) and a short lifetime acceptor (A).

[0026]FIG. 12. Intuitive description of resonance energy transfer from ahigh quantum yield donor (Q_(D)=1.0, top) and a low quantum yield donor(Q_(D)=0.1, bottom). For both panels ε_(A)/ε_(D)=0.1. For the lowquantum yield donor RET results in an increase in the overall quantumefficiency of the tandem luminophore.

[0027]FIG. 13. Chemical structures of the donors and acceptors used inthis report.

[0028]FIG. 14. Emission spectra of the acridine orange donor [Donor]=5μM bound to DNA in the presence of the acceptors nile blue (top), TOTO-3(middle), or TO-PRO-3 (bottom). The inserts show the acceptor regionwith the amplitude increased by a factor of 40. The dashed lines showthe emission of acceptor-alone with DNA, but without donor, at thehighest acceptor concentration used in the figure. The donor is presentat 1 donor per 200 base pairs. The number for the donor-acceptor (DA)pair is the number of base pairs for each acceptor.

[0029]FIG. 15. Emission spectra of ethidium bromide donor [Donor]=10 μMbound to DNA in the presence of the acceptors nile blue (top), TOTO-3(middle) or TO-PRO-3 (bottom). The dashed lines show the emission of theacceptor-alone with DNA, but without donor, at the highest used acceptorconcentration. The donor is present at 1 donor per 100 base pairs. Thenumber for the donor-acceptor (DA) pair is the number of base pairs foreach acceptor.

[0030]FIG. 16. Emission spectra of Ru-BD([Ru(bpy)₂dppz]²⁺) donor[Donor]=20 μM bound to DNA in the presence of the acceptors nile blue(top), TOTO-3 (middle) and TO-PRO-3 (bottom). The dashed lines show theemission spectra of the acceptor-alone with DNA, but without donor, atthe highest used acceptor concentration. One donor is present per 50base pairs. The number for the donor-acceptor (DA) pair is the number ofbase pairs for each acceptor.

[0031]FIG. 17. Uncorrected excitation spectra of Ru-BD and TO-PRO-3bound to DNA (- - -), and of TO-PRO-3 alone bound to DNA (-). The lowerpanel shows the ratio of the two excitation spectra.

[0032]FIG. 18. Transmission spectra of the emission filters used formeasuring the frequency-domain intensity decays (-). The dashed linesshow representative emission spectra of the donor alone (D) and donorplus acceptor (DA) samples.

[0033]FIG. 19. Frequency-domain intensity decays of Ru-BD bound to DNAin the absence and presence of the nile blue acceptor. The solid dotsrepresent the phase or modulation values and the solid lines the bestmulti-exponential fits to the data. In the middle and lower panels thedotted lines represent the donor-alone and acceptor-alone frequencyresponses, respectively.

[0034]FIG. 20. Frequency-domain intensity decay of Ru-BD bound to DNA inthe absence and presence of the TOTO-3 acceptor. See legend to FIG. 7.

[0035]FIG. 21. Frequency-domain intensity decay of Ru-BD bound to DNA inthe absence and presence of the TO-PRO-3 acceptor. See legend to FIG. 7.

[0036]FIG. 22. Time-domain intensity decay of Ru-BD and acceptorcomplexes with DNA.

[0037] Merely by way of example, the invention is illustrated by thetandem luminophore shown in FIG. 1. This luminophore displays resonanceenergy transfer (RET) from the exemplary ruthenium metal-ligand complex(MLC) donor shown to the exemplary Texas red (TR) acceptor. The termluminophore is used because emission from these particular MLCs displayboth singlet and triplet character. In no way is this term to limit thisinvention. A metal ligand complex is used as the donor because thetransition from the triplet excited state to the singlet ground state isnot allowed and these molecules display long lifetimes ranging from 100ns to 10 μs [15-17]. Some MLCs are known which display still longerdecay times from 50 to 260 μs [18-20]. Because of the long lifetimes,ease of synthesis, and range of spectral properties, the MLCs have beendeveloped as luminescent probes in physical, analytical and biophysicalchemistry [21-28].

[0038] While the MLCs display some favorable spectral properties, otherproperties are less favorable. For example, the MLCs display lowextinction coefficients, typically less than 20,000 M⁻¹ cm⁻¹, e.g., near10,000 M⁻¹ cm⁻¹; which is one reason for the long decay times [14], butwhich results in decreased sensitivity. Additionally, most MLCs displaylow quantum yields which rarely exceed 0.1, and the quantum yields ofthe MLCs with the longest decay times are often smaller [18-20].Finally, the emission spectra are broad, which makes it more difficultto quantify the MLC emission in the presence of autofluorescence becausethe background is also widely distributed across the wavelength scale.Broad emission spectra also result in significant spectral overlap ofthe emission spectra of various MLCs, and an inability to usemeasurements at multiple emission wavelengths to resolve multiplespecies in a macroscopic or microscopic samples.

[0039] In the present invention, these limitations of the available MLCand red/NIR probes are overcome. The luminophore of this inventioncomprises a MLC which displays a long lifetime and low quantum yield andwhich is, e.g., covalently linked to a high quantum yield acceptor whichtypically is a short lifetime fluorophore. The luminophore is excited ata wavelength where the MLC absorbs, typically near 450 nm for theexemplary ruthenium (Ru) MLCs. The emission therefrom is red shifted tolonger wavelengths by RET to the red/NIR emitting acceptor. Some longwavelength probes have low absorption near 450 nm so that most of theincident light is absorbed by the donor. Much if not most of theacceptor emission is thus due to energy transfer from the MLC.

[0040] Following pulsed excitation, the excited state population of theMLC becomes the only excitation source for the acceptor, which continuesto emit as long as MLCs remain in the excited state. Such luminophorescan still display long decay times in the presence of RET. For instance,if the MLC donor displays a lifetime of 1 μs in the absence of RET, thelifetime of the luminophore is expected to decrease to 100 ns if the RETefficiency is 90%, e.g., D-A distance being 0.7 Ro (Förster distance). Adecay time of 100 ns is much longer than can be obtained with knownred/NIR probes and 100 ns is longer than most autofluorescence. With a10 μs decay time donor, 90% transfer efficiency will result in a 1 μscomponent in the acceptor decay.

[0041] Assuming that the acceptor does not absorb at the donorexcitation wavelength (λ_(D) ^(ex)), the acceptor is excited solely byRET from the donor. Since the acceptor lifetime is short (τ_(D)=1 ns),the acceptor intensity will closely follow the donor intensity. Hencethe acceptor will display the same decay time as the donor and theacceptor decay time (τ_(AD)) will be near 100 ns. Most acceptors willdisplay some absorption at the donor excitation wavelength. In this casethe acceptor emission will typically display two decay times, a nscomponent due to directly excited acceptor, and long decay time near 100ns due to RET from the donor. The long lifetime emission acceptor can bereadily isolated with gated detection, which is readily accomplishedwith photo multiplier tubes (PMTs) [78-80]. Gated detection isfrequently used in immunoassay based on the lanthanides [81, 82].

[0042] An important advantage of such a RET probe (FIG. 11) is anincrease in the effective quantum yield of the long lifetimeluminophore. This effect is illustrated in FIG. 12. Suppose the donorand acceptor both display quantum yields of unity (Q_(D)=Q_(A)=1.0). Inthis case (top) RET quenches the donor and results in an equivalentincrease in the emission intensity of the acceptor. The integrated ortotal intensity of the donor and acceptor remains the same in thepresence or absence of RET.

[0043] A surprisingly different result is obtained if the donor displaysa low quantum yield. For example, the commonly used ruthenium MLCs havequantum yields of 0.05 or less. In this case the donor emission withoutRET is much weaker (FIG. 12, lower panel). However, RET to a nearbyacceptor still results in the same increased intensity of the acceptor.More specifically, the transfer efficiency can approach unity eventhough the donor quantum yield is low. A favorable result of efficientRET from the donor is that the wavelength integrated intensity of theD-A pair can be almost 20-fold larger than that of the donor or acceptoralone. More specifically, for 100% transfer efficiency, the overallquantum yield becomes the quantum yield of the acceptor. Theseconsiderations suggest that tandem RET probes based on MLC donors can beused to create long lifetime probes, with red-NIR emission, with theadded advantage of an increased effective quantum yield. Additionally,the modular design of these probes allows practical and rationaladjustment of the spectra properties including the excitation andemission wavelengths and the decay times.

[0044] Luminophores of this invention are typified in FIG. 11, whichshows a long lifetime donor (D) which is covalently linked to anacceptor (A), with spectral properties such that resonance energytransfer occurs with moderate to high efficiency. In this case theD-to-A distance is assumed to be 0.7 R₀, where R₀ is the Försterdistance,. This separation results in approximately 90% transfer. Thedonor is preferably a luminescent transition metal-ligand complex (MLC).Many such MLCs are known, and they can display a wide range ofabsorption and emission wavelengths and long decay times ranging from100 ns to 10 μs [15-16]. In recent years these complexes have beendeveloped for use as luminescent probes [21, 22] for studies of proteindynamics, immunoassays and chemical sensing [23-28].

[0045] The theory and application of RET have been described in numerousreviews [31-33]. (The following discussion of theory is in no wayintended to be limiting.) Discussed here are those aspects of RET neededto demonstrate the occurrence of a RET enhanced quantum yield and theappearance of a long lifetime component in the acceptor decay. The rateof energy transfer for a donor to an acceptor is given by$\begin{matrix}{k_{T} = {\frac{1}{\tau_{D}^{0}}\left( \frac{R_{0}}{r} \right)^{6}}} & (1)\end{matrix}$

[0046] where τ_(D) ⁰ is the donor lifetime in the absence of acceptor, ris the donor-to-acceptor distance, and R₀ is the Förster distance atwhich RET is 50% efficient. The value of R₀ can be accurately calculatedfrom the spectral properties of the donor and acceptor.

[0047] Consider the donor-acceptor pair FIG. 1. Assume the donor has alifetime τ_(D) ⁰=1 μs and the acceptor a lifetime of τ_(A) ⁰=1 ns whendirectly excited. The efficiency of energy transfer is given by theratio of the transfer rate to the total rate of donor deactivation,which is the reciprocal of the lifetime. Hence the transfer efficiency(E) from the donor is given by $\begin{matrix}{E = {\frac{k_{T}}{k_{T} + \Gamma_{D}} = \frac{k_{T}}{\lambda_{D} + k_{D} + k_{T}}}} & (2)\end{matrix}$

[0048] where Γ_(D)=(τ_(D) ⁰)⁻¹=(λ_(D)+k_(D))⁻¹ is the decay rate of thedonor in the absence of acceptor, and λ_(D) and k_(D) are the radiativeand non-radiative decay rates, respectively (FIG. 2). The transferefficiency (E) can be determined experimentally from the relativeintensities of the donor in the absence (F_(D)) and presence (F_(DA)) ofacceptor $\begin{matrix}{E = {1 - \frac{F_{DA}}{F_{D}}}} & (3)\end{matrix}$

[0049] The transfer efficiency can also be determined from the donordecay times in the absence (τ_(D) ⁰) or presence (τ_(D)) of acceptors$\begin{matrix}{E = {1 - \frac{\tau_{D}}{\tau_{D}^{0}}}} & (4)\end{matrix}$

[0050] This expression is only valid when the donor decay is a singleexponential. The decay time of the donor in the presence of acceptor isgiven by

τ_(D)=1/(k _(T)+Γ_(D))  (5)

[0051] which is the reciprocal of the sum of the deactivation rates ofthe donor.

[0052] The possibility of using rapid RET to improve the system quantumyield with low quantum yield donors can be seen from the equations whichdescribe the donor (F_(D)) or acceptor (F_(A)) intensities. In thekinetic scheme of FIG. 2, the intensity of the donor and acceptor isproportional to the amount of light absorbed or the extinctioncoefficient (ε_(D) and ε_(A)) and the fraction of the absorbed lightwhich is emitted. Hence in the absence of RET $\begin{matrix}{F_{D}^{\circ} = {\frac{\lambda_{D}ɛ_{D}}{\lambda_{D} + k_{D}} = {{Q_{D}^{0}ɛ_{D}} = {\tau_{D}^{0}\lambda_{D}ɛ_{D}}}}} & (6) \\{F_{A}^{\circ} = {\frac{\lambda_{A}ɛ_{A}}{\lambda_{A} + k_{A}} = {{Q_{A}^{0}ɛ_{A}} = {\tau_{A}^{0}\lambda_{A}ɛ_{A}}}}} & (7)\end{matrix}$

[0053] where ε_(A) and ε_(D) are the extinction coefficients at thewavelength used to excite the donor. The lifetimes of the unquencheddonor and the directly excited acceptor are given by (τ_(D)⁰)⁻¹=λ_(D)+k_(D) and (τ_(A) ⁰)⁻¹=λ_(A)+k_(A). The quantum yields of thedonors or acceptors in the absence of energy transfer are given by theratio of the emissive rates (λ_(D) or λ_(A)) to the sum of the rateprocess which depopulates the excited state (λ_(D)+k_(D)) or(λ_(A)+k_(A)). There is usually some acceptor emission even in theabsence of RET due to direct absorption (excitation) of the acceptorresulting from the non-zero value of ε_(A). For clarity theproportionality constant is dropped which should be on the right side ofeach equations 6 and 7.

[0054] In the absence of RET the total intensity (F_(T) ^(o)) of thedonor (F_(D) ^(o)) and acceptor (F_(A) ^(o)) is that due to directexcitation of both species $\begin{matrix}{F_{T}^{\circ} = {{F_{D}^{\circ} + F_{A}^{\circ}} = {{\frac{\lambda_{D}ɛ_{D}}{\lambda_{D} + k_{D}} + \frac{\lambda_{A}ɛ_{A}}{\lambda_{A} + k_{A}}} = {{Q_{D}^{0}ɛ_{D}} + {Q_{A}^{0}ɛ_{A}}}}}} & (8)\end{matrix}$

[0055] where F_(T) ^(o) is the total emission in the absence oftransfer. Now assume RET occurs with a rate k_(T). The intensities ofthe donor and acceptor are given by $\begin{matrix}{F_{D} = {\frac{\lambda_{D}ɛ_{D}}{\lambda_{D} + k_{D} + k_{T}} = {Q_{D}ɛ_{D}}}} & (9) \\{F_{A} = {\frac{\lambda_{A}ɛ_{A}}{\lambda_{A} + k_{A}} + {\frac{k_{T}ɛ_{D}}{\lambda_{D} + k_{D} + k_{T}} \cdot \frac{\lambda_{A}}{\lambda_{A} + k_{A}}}}} & (10)\end{matrix}$

[0056] The intensity or quantum yield of the donorQ_(D)=λ_(D)/(λ_(D)+k_(D)+k_(T)) is decreased by an additional rate k_(T)which depopulates the donor (eq. 9). The intensity of the acceptor isincreased by the transfer rate k_(T). The transfer efficiency termE=k_(T)/(λ_(D)+k_(D)+k_(T)) in eq. 10 can be understood as the fractionof absorbed photons absorbed by the donor which are transferred to theacceptor. These transferred photons are emitted with a quantum yieldQ_(A)=λ_(A)/(λ_(A)+k_(A)). The energy received from the donor is emittedwith the quantum yield of the acceptor. The combined emission intensityof the donor and acceptor is given by

F _(T) =F _(D) +F _(A) =Q _(D)ε_(D) +Q _(A) ^(o)(ε_(A) +Eε _(D))=Q _(D)⁰ε_(D)(1−E)+Q _(A) ⁰(ε_(A) +Eε _(D))  (11)

[0057] It is instructive to consider the limits of very slow (k_(T)→0and E→0) and very fast (k_(T)∞) energy transfer. In the limit of noenergy transfer the total intensity becomes equal to that of a mixtureof two non-interacting fluorophores (eq. 8). In the limit of rapidtransfer (k_(T)→∞ and E→1) the total intensity becomes $\begin{matrix}{F_{T} = {\frac{\lambda_{A}\left( {ɛ_{A} + ɛ_{D}} \right)}{\lambda_{A} + k_{A}} = {Q_{A}\left( {ɛ_{A} + ɛ_{D}} \right)}}} & (12)\end{matrix}$

[0058] This is an important result which indicates the total intensityis proportional to the sum of the extinction coefficients and to thequantum yield of the acceptor. This occurs because the energy transfercan occur with an efficiency of one even if the donor quantum yield islow. If the rate of energy transfer is fast and if the acceptor absorbsweakly the excitation wavelength (ε_(A)<<ε_(D)) then $\begin{matrix}{F_{T} = {\frac{\lambda_{A}ɛ_{D}}{\lambda_{A} + k_{A}} = {Q_{A}ɛ_{D}}}} & (13)\end{matrix}$

[0059] This equation shows that with rapid energy transfer and nodirectly excited acceptor the acceptor emission intensity isproportional to the amount of light absorbed by the donor and thequantum yield of the acceptor. The donor-acceptor pair becomes essentialto a new fluorophore with an extinction coefficient E_(D) and a quantumyield Q_(A).

[0060] It is informative to consider the time-dependent decays of thedonor, acceptor and the total emission. These expressions are similar tothose known for an excited state reaction [34-37]. Here, the reversetransfer rate from A to D is zero (FIG. 2). Additionally, since bothdonor and acceptor are present all times, there is some directexcitation of the acceptor in addition to the acceptor which is excitedby RET from the donor. The time-dependent changes in the donor andacceptor populations are given by $\begin{matrix}{\frac{\lbrack D\rbrack}{t} = {{- {\left( {\Gamma_{D} + k_{T}} \right)\lbrack D\rbrack}} + {ɛ_{D}{L(t)}}}} & (14) \\{\frac{\lbrack A\rbrack}{t} = {{- {\Gamma_{A}\lbrack A\rbrack}} + {k_{T}\lbrack D\rbrack} + {ɛ_{A}{L(t)}}}} & (15)\end{matrix}$

[0061] where L(t) is the excitation function. The square brackets aretaken to indicate the excited state population of each species. Thetime-dependent decays of the donor and acceptor are given by

I _(o)(t)=N _(D) ⁰ exp[−Γ_(D) +K _(T))t]  (16)

I _(A)(t)=A exp[−Γ_(D) +k _(T))t]−(N ^(o) _(A) −A) exp[Γ_(A) t]  (17)

[0062] where N_(D) ⁰ and N_(A) ⁰ are the number of excited donors andacceptor molecules at t=0. The pre-exponential factors in eqs. 16 and 17are proportional to ε_(D)L(t) and ε_(A)L(t), respectively, but notshown. The factor A $\begin{matrix}{A = {\frac{N_{D}^{0}k_{T}}{\Gamma_{A} - \Gamma_{D} - k_{T}} = \frac{{- N_{D}^{0}}k_{T}}{\Gamma_{D} - \Gamma_{A} + k_{T}}}} & (18)\end{matrix}$

[0063] depends on the efficiency by which the acceptor is pumped by thedonor. According to equation 16, the donor decay I_(A) (t) is the usualdecay rate of a donor with a transfer rate k_(T). The acceptor decaycontains a component with the lifetime of the acceptor τ_(A)

0 and a component with the lifetime of the quenched donor τ_(D).

[0064] Suppose the acceptor decay is very rapid, that is, the directlyexcited acceptor displays a short lifetime, τ_(A)

⁰ 6 0 or Γ_(A) is very large. Then the acceptor decay becomes

I _(A)(t)=A exp[−(Γ_(A) ⁻ +k _(T))t].  (19)

[0065] This result shows that in the limit of a short acceptor lifetimethe acceptor emission resulting from energy transfer displays the samelifetime as the quenched donor. A similar result is shown if one assumesτ_(D)>>τ_(A) or Γ_(A)>>Γ_(D). In this case the rightmost term inequation 17 decays rapidly to zero, relative to the donor decay, and theacceptor decay resulting from RET displays the same decay time as thedonor. If there are no initially excited acceptors, N_(A) ⁰=0, equal andopposite pre-exponential factors are obtained and the acceptor decaysaccording to

I _(A)(t)=A exp[−(Γ_(D) +k _(T))t]−A exp[Γ_(A) t]  (20)

[0066] Moreover, the inventor's publication, Lakowicz et al., AnalyticalBiochemistry 288, 62-75 (2001) is entirely incorporated by referenceherein.

[0067] In one aspect, this invention thus involves the increase of theeffective quantum yield of a luminophore by rapid RET in long lifetimeMLC components having low quantum yields. Such an increase in effectivequantum yield has not previously been important in the biochemical usesof RET [29-30 and 38-46] because most organic donors have good quantumyields. The increased effective quantum yield of the donor has not beenimportant for RET with, e.g., the lanthanides because transfer from theorganic chelates to the lanthanides is efficient, and the shieldedlanthanide donors often display quantum yields near unity [42-46]. (Seealso the enhancement of lanthanide emission when bound to essentialnon-luminescent DNA or nucleotides [47-49]). There are numerous primaryreports and review articles on RET, and the concept of using theacceptor emission to measure the transfer efficiency is not new [38-41].Additionally, Selvin and co-workers have already noted the usefulness ofmeasuring the long lifetime acceptor emission with lanthanide donors toselectively detect D-A pairs [42] and to provide a long decay time forthe acceptor [43, 44]. Donors and acceptors with short decay times havebeen covalently linked for use in DNA sequencing [30, and Ju et al.PNAS, USA, 92, 4347 (1995)] and as high affinity dyes which bindnon-covalently to DNA [45, 46].

[0068] The approach of this invention to tandem luminophores can berationally and routinely used to obtain the desired spectral properties.RET is a highly predictable phenomena. The long acceptor decay time canbe increased by a longer spacer. Less spectral overlap of the D and Acan be obtained using shorter wavelength rhenium MLC donors or longerwavelength acceptors.

[0069] These tandem luminophores can be prepared in conjugatable formsand used as a single reagent. This invention can also be applied to themeasurement of protein or DNA association reactions where the donor andacceptor are present in separate molecules and are placed in closeassociation by the interactions of the separate molecules.

[0070] The luminophores of this invention can be used as labels fullyanalogous to prior art labels, e.g., those discussed in the referencescited herein, e.g., by conventional covalent linking to desiredmolecules to be detected, e.g., nucleic acid proteins, cells, etc.,probes based thereon etc.

[0071] Thus, this invention involves donor molecules/portions, D,typically having low quantum-yields less than about 0.2 or even lower,e.g., about 0.1 or about 0.01-0.2, 0.1-0.2, etc. Such donor moleculesare well known. Typically they are metal ligand complexes, of transitionmetals (e.g., atomic numbers 21-30, 39-48 and 72-80); those of thelanthanides (e.g. atomic numbers 57-71, 81-83) are also possible, butthese typically have high quantum yields. A wide variety of well knowndonor-type metal ligand complexes are well known. See references 15-27.See, as well, Demas et al., Coordination Chemistry Reviews 211 (2001)317-351; Stufkens et al., Coordination Chemistry Reviews 177 (1998)127-179. Typically, but not in a limiting way, these are of the di-iminee.g., bipyridyl type. Most preferred are the transition metal complexes,especially those of renium, ruthenium, osmium and iridium. Such Dmolecules are well known as having low quantum yields and having broademission spectra at relatively long wavelengths, as mentioned above.Their emission life times are also relatively long as also mentionedabove.

[0072] The acceptor molecules/portions are also per se well known in thefield. Typically, these are dye molecules such as Texas Red. Albumin 633or 670, CY5, fluorescein dyes, polymethine dyes, cyanine dyes,squarilium dyes, croconium dyes, merocyanine dyes, oxonol dyes, and manyothers. See e.g., WO 98/22146; and topics in Fluorescence Spectroscopy,Vol. 4: Probe Design and Chemical Sensing, ed. Joseph R. Lakowicz,Plenum Press, N.Y., 1994, Chapter 6, R. B. Thompson, pp. 151-182, andChapter 7, Guillermo A. Casay, et al., pp. 183-222. These acceptormolecules are known as having high quantum yields per se and as emittingin relatively long wavelength regions with long lived decay times.

[0073] This invention provides a combination of molecules or closelyassociated component species involving both D and A molecules/portionse.g., covalently linked to one another or in close association with eachother such that the spacing of the two molecules, in all cases, iseffective for resonant energy transfer from the donor to the acceptor.This may be achieved not only by covalent linking but also by use ofconventional biological association reactions, e.g., nucleic acidhybridization between two nucleic acid molecules (DNA, RNA, etc.), onebonded to the donor and the other bonded to the acceptor. Suchassociation can also be achieved by other similar specificallyinteracting molecules, e.g., protein/nucleic acid, antibody/antigen,receptor/ligand, etc. Details of the linking of the donor and/oracceptor molecules/portions to any such molecules are fullyconventional.

[0074] Where a D/A molecule is to be employed, the D portion is linkedto the A portion by a spacer or linker molecule, L. The nature of thespacer is non-critical, the effective parameter being the distancebetween D and A and the covalently linked combination. Thus, any of thewell known spacer molecules can be employed, e.g., polyalkylenemoieties, polyamino acid moieties (e.g., polyproline moieties of theexamples), maleimido moieties, isothiocyanate moieties, esters, ethers,secondary and tertiary amines, amides, the structures cited below, etc.

[0075] See any of the well known prior art linker-related disclosures inthis regard. In general, the closer D and A are spaced from each otherthe faster and more efficient will be the resonant energy transfer,e.g., as can be seen from the examples. Determination of an optimaldistance and a corresponding spacer is fully routine as can be seen fromthe literature cited herein. Typically, spacings are desired which willachieve transfer efficiencies about 10%-90%, e.g., 20-80%, 30-70%,40-60%, efficiencies around 50% typically being satisfactory. If thetransfer efficiency is too high, then the decay times achieved will betoo short.

[0076] As can be seen, by routine selection of the D-moiety, A-moietyand spacer distance, “designer” probes can be achieved in accordancewith this invention. See, e.g., Stufkens et al., above, e.g.,pp.171-174; Chen et al., J. Am. Chem. Soc. 2000, 122, 657-660.Typically, the resultant long wavelength emission will be in the rangeof 400-1200 nm, e.g., 450-1200, 550-1000 nm and more typically 600-900nm. Decay life times (half lives) will typically be greater than 25 ns,typically 25 ns-100 μ, more typically 50 ns-10 μs, and most typically 50ns-2 μs. Luminophores of this invention having a desired emissionwavelength and lifetime can be prepared in accordance with well knownconsiderations and the guidance provided by this specification.Selection of the A and D moieties appropriate for a desired emissionwavelength range can be made using conventional considerations e.g., asdiscussed in references 15-27, e.g., by suitable routine selection ofmetal and ligand combinations. Modification of the spacing lengthbetween D and A will similarly routinely be achievable by appropriateselection of chemical linking moieties, to achieve a resultant desiredtransfer efficiency and life time.

[0077] The production of the D and A compounds according to theinvention can be carried out by conventional modification of thesubstances, which contain functionalities that can be coupled (e.g.,carboxyl, amino, and hydroxyl groups), according to processes well knownto one skilled in the art.

[0078] The production of the adducts according to the invention iscarried out by reaction of the dye with a metal ligand complex or ligandcomplex (followed by metallation) according to methods that are wellknown in the literature. The dyes and complexes must have reactivegroups that can be coupled in this regard or they must routinely beactivated in-situ or in advance by generation of these groups. Withregard, e.g., to amino- and sulfhydryl groups suitable reactive groupsare, for example, N-hydroxysuccinimidylester,N-hydroxy-succinimidylester-3-sulfate, isothiocyanates, isocyanates,maleimide-, haloacetyl, vinylsulfone groups. The coupling is preferablycarried out in an aqueous medium. In this case, the degree ofconcentration can be routinely controlled by stoichiometry and reactiontime. See e.g., Snyth. Commun. 23 (1993) 3078-94, DE-OS 3912046, CancerImmunol. Immunother. 41 (1995) 257-263, Cancer Research 54 (1994)2643-49.

[0079] Thus, as can be seen, this invention provides luminiphor probesemitting long wavelength radiation with high quantum yield despite theinvolvement of absorbing donors having low quantum yields. As a result,emitter probes are provided at wavelengths to which skin is at leasttranslucent, in which wavelength ranges background autofluorescence andnatural fluorophore emissions are minimized. Such long lifetime emissionis achieved also despite the use of acceptor portions (dyes) per sehaving short life times. This represents another significant advantagesince extant background fluorescence tends to be of significantlyshorter lifetimes than that achieved by the emitters of this invention.

[0080] The closely associated D/A pairs of this invention can be usedstraightforwardly in any of the usual probe-based techniques mentionedherein, e.g., including nucleic acid sequencing, hybridization assays,immunoassays, etc. This aspect is fully conventional. See e.g., Ota etal., Nucleic Acid Research, 1998, Vol. 26, No. 3, 735-743; Peterson etat., J. Am-Chem. Soc., 2000, 122, 7837-7838; Paris et al. Nucleic AcidResearch, 1998, Vol. 29, No. 16, 3789-3793; Templeton et al., Clin.Chem. 37/9, 1506-1512 (1991); Weissleder et al., Nature BiotechnologyVol. 17, April 1999, 375-378; Xiav et al., Proc. Natl. Acad Sci.,95,15309-15314, December 1998.

[0081] Another application of this invention is for the study ofmacromolecular association reactions, such as protein-proteininteractions, DNA hybridization [58-60], fluorescence in-situhybridization (FISH) [61], or the use of molecular beacons [62, 63]. Asan example, suppose it was necessary to test for binding ofdonor-labeled oligonucleotides to a mixture of acceptor-labeledoligonucleotides. When using a RuMLC donor and one of the acceptors usedin this report, most of the species labeled with donor or acceptor alonewill display little emission. In contrast the D-A pairs due tomacromolecular association will be brightly fluorescent. Additionally,the acceptor emission will be long lived. Using time-gated detectionbrightly fluorescent spots may become apparent against background ofweakly stained chromatin and/or short decay time. These spectralproperties will be useful for detection of oligonucleotide hybridizationon DNA arrays [64-65]. Such arrays are becoming widely used for analysisof gene expression [66-68].

[0082] Thus, a generic approach to obtaining an unusual combination ofspectral properties by using an appropriate D-A pairs is provided. Thisapproach can be used to create D-A pairs which acts as a singleluminophore, or this effect can be used to detect interactions insamples containing species labeled with the donor or acceptor. Thisapproach will also be useful in studies of macromolecular folding asillustrated by the use of RET to study ribozyme structures [69, 70]. Onecan also provide long lifetime donors linked to pH, Ca²⁺, or otheranalyte-sensitive fluorophores [71, 72]. If the analyte sensitivefluorophore displays distinct emission spectra with and without boundanalyte, then there will be a long lived component in the emission withthe spectral characteristics of each form. Finally, the use of theenhanced emission, and inhibition of the enhancement, can be used inmacromolecular binding assays in high throughput screening [73, 74].There appear to be numerous applications of our approach in biochemicaland biomedical research. ABBREVIATIONS A acceptor D donor D-Adonor-acceptor pair MLC metal-ligand complexes NIR near infrared PMTphotomultiplier tube TR Texas Red bpy 2,2′-bipyridine phen1,10-phenanthroline RET resonance energy transfer Ru Ru(bpy)₂(phen-ITC)which has been covalently linked to a peptide or DNA oligomer

[0083] In the foregoing and in the following examples, all temperaturesare set forth uncorrected in degrees Celsius; and, unless otherwiseindicated, all parts and percentages are by weight.

[0084] The entire disclosures of all applications, patents andpublications, cited above and below are hereby incorporated byreference.

EXAMPLE 1

[0085] Simulations were performed to predict the spectral properties ofthe D-A pair for typical decay times and quantum yields. For thesesimulations, eq. 11 was modified to use the normalized extinctioncoefficient ε′_(D) and ε′_(A)

[0086] where

Q _(T) =Q _(D) ⁰ε′_(D)(1−E)+Q ⁰(ε′_(A) +Eε′ _(D))  (21)

ε′_(D)=ε_(D)/(ε_(D)+ε_(A))  (22)

[0087] and

ε′_(A)=ε_(A/(ε) _(D)+ε_(A))  (23)

[0088]FIG. 3 shows the total quantum yield expected for three D-A pairsfor various transfer efficiencies. The quantum yield of the acceptor wasassumed to be high Q_(A) ⁰=0.9 (top), intermediate Q_(A) ⁰=0.5 (middle)and low Q_(A) ⁰=0.1 (lower panel). Since most acceptors will absorb atthe donor excitation wavelength, we assumed the normalized extinctioncoefficient of the acceptor was ε′_(A)=ε_(A)/(ε_(A)+ε_(D))=0.10. As thetransfer efficiency increases the total quantum yield approaches that ofthe acceptor. If the acceptor quantum yield is low (lower panel), thenenergy transfer decreases the overall quantum yield. Importantly, if thequantum yield of the acceptor is high (upper panel), the overall quantumyield approaches that of the acceptor for high transfer efficiency.

[0089] The intensity decays expected for the donor and acceptor in D-Apairs for various transfer efficiencies (FIG. 4) were also simulated.The assumed decay times were τ_(D) ⁰=1000 ns and τ_(A) ⁰=10 ns. Animportant conclusion from these simulations is that the acceptor candisplay long decay times. If the transfer efficiency is 33% (FIG. 4, toppanel), the acceptor shows a decay time with τ=667 ns (Table I). Thetransfer efficiency can be as high as 90.9% and the acceptor stilldisplay a 91 ns decay time. Thus, usefully long decay times can beobtained even with high transfer efficiency.

EXAMPLE 2

[0090] The practical usefulness of the tandem luminophores of thisinvention were demonstrated using the covalently linked D-A pairs shownin FIG. 1. These D-A pairs can be considered to be the probe or reagent,in the same manner that linked DNA pairs have been developed for DNAsequencing [29-30]. Alternatively, this unique long lifetime highquantum yield emission can be the result of protein or nucleic acidassociation reactions.

[0091] The Texas Red iodoacetamide with a C5 linker was purchased fromMolecular Probes, Inc. The [Ru(bpy)₂ (amino phenanthroline)]²⁺ was agift from Dr. Jonathan Dattelbaum. It was converted into isothiocyanateby treating with 500 μl of thiophosgene in 1 ml acetone for 3 hrs. Boththe solvent and thiophosgene were removed under a stream of nitrogen andthe isothiocynate was used immediately.

[0092] The oligo proline peptides with a cysteine at C-terminus weresynthesized at the biopolymer facility of University of Maryland Schoolof Medicine, Baltimore. The crude peptide was purified by RP-HPLC on aC18 column using a 0.1% TFA and 100% acetonitrole containing 0.05% TFA.The molecular weights were confirmed by mass spectroscopy.

[0093] The peptides were labeled first with the acceptor. Typically a mMsolution of the peptide in 0.2 M bicarbonate buffer, pH 8.5, was reactedwith a 2-fold excess iodoacetamide for 6 hours. The resulting peptidewas purified from the free probe using a column of Sephadex G-15 runningin 20% DMF solution. The labeled peptide was further purified by HPLC.

[0094] To prepare the double labeled peptide the acceptor labeledpeptide was further reacted with a five-fold excess Ru isothiocynate in0.2 M bicarbonate, pH 9.0 for 6 hours. The peptide was separated fromthe free probe by passing through a Sephadex G-15 column and furtherpurified on HPLC. To prepare the donor-only peptide, the sulphydrylgroup was first blocked with a five-fold excess iodoacetic acid at pH8.5 for 1 hr and to same reaction mixture a five-fold excess of theisothiocyanate was added, the pH was adjusted to 9 and allowed to reactfor 6 hours. The free dye was separated on a Sephadex G-15 column andthe donor-labeled peptide was purified by HPLC. The purified peptideswere lyophilized and stored as water solutions at 4° C.

[0095] The steady-state measurements were done in an aqueous 5 mM hepes,100 mM NaCl, pH 8. The measurements in propylene glycol were withoutbuffer with the propylene glycol at least 98%, the remainder beingwater. For the steady-state measurements the peptide concentrations wereless than 2 μM and about 10 μM for the time-resolved measurements. Anaqueous solution of rhodamine B with a lifetime of 1.68 ns was used asthe reference. The frequency-domain lifetime measurements were done on aSLM instrument with a LED emitting at 450 nm as a light source. Theemission was observed through a 630/40 nm bandpass filter.

[0096] The emission spectra of Ru-(pro)₆-cys-TR (FIG. 1), referred to asthe (pro)₆ D-A pair was examined. As a control for the donor-alone (D),the structure shown in FIG. 1 was used with the sulfhydryl group blockedwith iodoacetamide. For the acceptor (A), the structure shown in FIG. 1was used without the covalently linked donors. Emission spectra of thesethree compounds are shown in FIG. 5. These spectra were obtained usingthe same molar concentrations of D, A and D-A. The overall intensity ofthe D-A pair is about 5-fold larger than the sum of the donor andacceptor alone. This result demonstrates that a tandem luminophore witha low quantum yield donor can display a higher quantum yield than eitherspecies alone.

[0097]FIG. 6 shows the absorption and excitation spectra of D, A andD-A. The absorption spectra of D-A was found to be essentially identicalto the sum of the D-alone and A-alone absorption spectra (top).Contrasting results were found for the excitation spectra (FIG. 6,bottom). In this case the intensity of the long wavelength emission withexcitation at 450 nm is about 6-fold greater than that of the directlyexcited acceptor and about 20-fold larger than the donor alone. Thisresult also demonstrates the role of energy transfer in increasing theeffective quantum yield of the donor.

[0098] The enhanced emission demonstrated in FIGS. 5 and 6 is determinedby the relative extinction coefficients of the donor and acceptor at theexcitation wavelength. The ratio of the donor to the absorption spectrais shown in the top panel of FIG. 7. This ratio displays a maximum near6 at 450 nm, which is near the peak of the donor absorption and theminimum of the acceptor absorption. The ratio of the excitation spectrashows the same trend, with a maximum near 450 nm (FIG. 7, bottom). Theseresults demonstrate that the enhancement at the acceptor emission isdetermined by the ratio of the light absorbed by each species.

[0099] Data also showed that the enhanced red emission could be obtainedwith usefully long decay times. This is an important considerationbecause if the donor and acceptor are too close, or the rate of transferis too fast, then the donor decay time will be shortened towards the nsvalue characteristic of the directly excited acceptor. Thefrequency-domain intensity decay of D, A and D-A are shown in water(FIG. 8, top) and in propylene glycol (bottom). For ease ofunderstanding, the frequency-domain data were used to reconstruct thetime-dependent decays (FIG. 9). In the absence of acceptor, thedonor-alone displays a mostly single exponential decay with a decay timeof 515 ns (top). The donor decay time is longer in propylene glycol(bottom), near 820 ns. The decay time of the directly excited acceptoris much shorter and near 4 ns in either solvent.

[0100] The D-A pair measured at the acceptor emission wavelengthdisplays a more complex intensity decay, as can be seen from thefrequency responses for D-pro₆-A (FIG. 8) or D-pro₈-A (FIG. 9). Theacceptor in D-pro₈-A displays a longer decay time as seen from the shiftto lower frequency of D-pro₈-A as compared to D-pro₆-A The reconstructedintensity decays are shown in FIG. 10 and the intensity decay parametersare summarized in Table II. For a D-A pair at a single distance, asingle decay time is expected for the donor. The heterogeneous decays ofthe D-A pairs is probably the result of a range of D-to-A distances dueto the flexibility of the linkers between hexaproline and the probes.There are 12 chemical bonds between the last proline and Texas Red. Whenusing an acceptor with a shorter linker, a more mono-exponential decaywill result. Nonetheless, the D-pro₆-A displays a long decay time near22 ns in water and 55 ns in propylene glycol. The components areassigned as due to the acceptors which are being excited by the exciteddonor population. A greater than 10-fold reduction in the donor decaytime due to RET is consistent with the greater than 90% RET efficiencyshown by this D-A pair.

[0101] Similar data were collected for the larger D-A pair with the pro₈spacer, D-pro₈-A (Table II). The frequency-domain data are shown in FIG.9 and the time-domain representations are shown in FIG. 10. For thismore widely spaced D-A pair the acceptor shows a decay time of 50 ns inwater and 130 ns in propylene glycol. Hence long decay times exceeding100 ns can be obtained using such tandem luminophores. TABLE I ExpectedLifetimes and Total Quantum Yields for D-A pairs^(a) AcceptorFluorescence Total Quantum Yield of the Transfer Efficiency LifetimesSystem E τ₁ [ns] τ₂ [ns] Q_(T) = Q_(D) + Q_(A) 0 10 — 0.108 0.091 10 9090.180 0.333 10 667 0.372 0.500 10 500 0.504 0.667 10 333 0.636 0.833 10167 0.768 0.909 10 91 0.829 0.950 10 48 0.860 0.980 10 20 0.884

[0102] a τ_(D) ⁰=1000 ns, τ_(A) ⁰=10 ns, Q_(D) ⁰=0.02, Q_(A) ⁰=0.90. Forthese calculations we assumed the extinction coefficient of the donor is9-fold larger than that of the acceptor, at the excitation wavelength.TABLE II Multi-exponential intensity decay analysis for the donor,acceptor and DA pairs shown in Scheme I^(a) Solvent/ Compound Water Qα_(i) ^(b) f_(i) τ_(i) χ_(R) ² D-pro₆ 0.0333 0.099 0.009 41.2 1.45^(c)0.901 0.991 516.7 pro₆-A 0.360 1.0 1.0 4.0 1.26 D-pro₆-A 0.33 0.4690.198 3.5 0.82 0.353 0.304 7.0 0.178 0.458 22.7 D-pro₈-A — 0.784 0.2874.4 0.137 0.252 22.3 0.079 0.461 71.1 Propylene Glycol D-pro-₆ — 0.1250.014 79.4 0.98 0.875 0.986 785 pro₆-A 1.0 1.0 4.1 2.36 D-pro₆-A — 0.8030.363 7.9 0.178 0.245 33.4 0.069 0.392 99.5 0.51 D-pro₈-A — 0.839 0.2255.1 1.3 0.089 0.170 36.3 0.072 0.604 157.8

EXAMPLE 3

[0103] Materials: CT-DNA, Tris.HCl and EDTA was obtained from Sigma (St.Louis, Mo.). Ru-BD was synthesized by the method described previously[51,52]. AO, EB, TOTO-3 and TO-PRO-3 were purchased from MolecularProbes (Eugene, Oreg.) and NB was from Aldrich (Milwaukee, Wis.). Allreagents were used without further purification and water was deionizedwith a Milli-Q system. To convert CT-DNA into linear fragmentscomparable in length to one persistent length, about 5 mg/ml solution ofCT-DNA was sonicated approximately 10 min while submerged in an icebath. The sonicated DNA solution was centrifuged for 1 hr at 75,000 ×gto remove titanium particles and undissolved DNA. All experiments wereundertaken at room temperature in 2 mM Tris.HCl, pH 8.0, containing 0.1mM EDTA.

[0104] Absorption and steady-state fluorescence measurement: AO, EB andRu-BD served as donors and NB, TOTO-3 and TO-PRO-3 were used asacceptors. About 5-10 mM stock solutions of AO, Ru-BD and NB wereprepared in dimethylformamide and about a 10 mM stock solution of EBwere made in DMSO. The final DMF concentration in all solutions was lessthan 1% (v/v). The concentration of DNA was quantified using a molarextinction coefficient of 13,300 M⁻¹ cm⁻¹ (expressed as bp) at 260 nm.The DNA concentration was 1 mM bp while the concentrations of AO, EB andRu-BD were 5, 10 and 20 μM, respectively. Concentration of the probeswere determined using the extinction coefficients in Table III. Thehighest acceptor concentrations of Ru-BD/NB, Ru-BD/TOTO-3, and Ru-BD/TOPRO-3 D-A pairs were 120, 60 and 120 μM, respectively. Because TOTO-3and TO-PRO-3 were supplied as 1 mM stock solutions in DMSO, the maximumpercentages of DMSO in the Ru-BD/TOTO-3 and Ru-BD/TO-PRO-3 D-A pairswere 6 and 12%(v/v), respectively. In preliminary experiments, we foundthat DMSO increased the steady-state fluorescence intensity of RuBD(data not shown). Hence, we added aliquots of DMSO to obtain 6 and12%(v/v) DMSO in all Ru-BD/TOTO-3 and Ru-BD/TO-PRO-3 D-A pairs,respectively, to equalize the effect of DMSO. UV-visible absorptionspectra were measured with a Hewlett-Packard 8453 diode arrayspectrophotometer with ±1 nm resolution. Steady-state fluorescencemeasurements were carried out using an Aminco SLM AB2 spectrofluorometer(Spectronic Instruments, Inc., IL) under magic angle conditions. Theexcitation wavelengths of AO, EB and RuBD were 470, 518 and 440 nm,respectively.

[0105] Frequency-domain fluorescence measurements: Measurements wereperformed using the instruments described previously [75] and modifiedwith a data acquisition card from ISS, Inc. (Urbana, Ill.) [76]. Theexcitation source was a blue LED LNG992CFBW (Panasonic, Japan) withluminous intensity of 1,500 mcd, and an LED driver LDX-3412 (ILXLightwave, Boseman, Mo.) provided 30 mA of current at frequencies from 1to 9.3 MHz. A 450RD55 interference filter (Omega Optical, Inc.,Brattleboro, Vt.) and a 4-96 color glass filter (Corning Glass Work,Corning, N.Y.) were used to isolate the excitation wavelength. RhodamineB in water was utilized as a lifetime standard. The transmission curvesof the filters for isolating the emission from the donor, D-A pairs, andacceptors are shown below (FIG. 18).

[0106] Steady State Spectra

[0107] DNA with non-covalently bound donors and acceptors was used totest the possibility of creating long lifetime luminophores with highquantum yields. Three donors, acridine orange (AO), ethidium bromide(EB) and [Ru(bpy)₂dppz]²⁺ (Ru-BD) were chosen. These structures areshown in FIG. 13. When bound in DNA the quantum yields decrease in thisrespective order (Table III). Acceptors, were nile blue (NB), TOTO-3 andTO-PRO-3 (FIG. 13), which display increasing quantum yields in thelisted order. Dyes non-covalently bound to DNA were used because thisapproach allowed us to select donors and acceptors with various quantumyields, without the need for chemical synthesis. Also, this approachallowed us to adjust the concentrations of donors and acceptors toobserve trends in the spectra. Based on the theory described above, thelargest overall increase in the total emission of the tandem luminophorewas expected to occur with RET between the lowest quantum yield donor(Ru-BD) and the highest quantum yield acceptor (TO-PRO-3).

[0108]FIG. 14 shows the emission spectra of AO bound to DNA withincreasing amounts of acceptor. With the high quantum yield AO donor theNB acceptor emission is almost undetectable (FIG. 14, top insert). Thequantum yield of the TOTO-3 acceptor is higher than that of NB, and thequantum yield of TO-PRO-3 is higher still. The acceptor emission becomesmore easily detectable as the acceptor quantum yields increase. In eachcase the observed acceptor emission is due to RET from the donor. Nosignificant acceptor emission was found for the acceptors bound to DNAin the absence of donor (dashed lines). An interesting aspect of FIG. 14is that RET from a high quantum yield donor (AD) to a low quantum yieldacceptor (NB) decreases the total emission from the donor and acceptor.

[0109]FIGS. 15 and 16 show emission spectra with the same acceptors, butwith EB and Ru-BD as the donors. Examination of these spectra shows thatthe enhancement of the acceptor emission is larger for Ru-BD than forEB. Also, the largest enhancements are seen for TO-PRO-3, the acceptorwith the highest quantum yield (FIG. 16, lower panel). In this case theacceptor emission is increased many-fold by energy transfer from theRu-BD donor. Also, the emission from the D-A system is considerablylarger than that of the donor alone bound to DNA, or the acceptor alonebound to DNA (dashed line). This effect is the opposite of that foundfor the AO/NB D-A pair. In this case the weakly fluorescent NB receivedmost of the energy by RET, but still emits with its own low quantumyield. For the Ru-BD/TO-PRO-3 D-A pair the strongly fluorescent TO-PRO-3receives most of the energy absorbed by the donor, in spite of the lowintrinsic quantum yield of the donor.

[0110] In the absence of energy transfer the intensity of the acceptoris proportional to ε_(A)Q_(A), where ε_(A) refers to the extinctioncoefficient of the acceptor at the donor excitation wavelength. Iftransfer is 100% effective the intensity of the acceptor is proportionalto (ε_(A)+ε_(D))/ε_(A). According to Table III this ratio is near 4.Examination of FIG. 16 (lowest panel) indicates that the acceptorenhancement is greater than 4, surprisingly. To further this effect theexcitation spectra of the D-A pair, and the acceptor alone, when boundto DNA were examined. On the same relative scale the acceptor alonedisplays essentially no emission upon excitation at 450 nm (FIG. 17).The lower panel shows the ratio of these excitation spectra, whichbecomes close to 40 at 450 nm. This ratio is larger than expected fromthe extinction coefficients listed in Table III. It appears thatexcitation of TO-PRO-3 near 450 nm results in less emission thanpredicted by its absorption spectrum. This effect could be due to thepresence of non-flourescent absorbing impurities, or absorption ofnon-fluorescent conformers of TO-PRO-3 at 450 nm. It is known that thisclass of dyes display weak fluorescence in water or when there istorsional motions about the central methine bridge [77]. Irrespective ofthe origin of this low intensity, the acceptor enhancement seen in FIG.16 is consistent with the excitation spectrum for this D-A pair.

[0111] Time-Resolved Decays

[0112] Frequency-domain intensity decays were measured through filtersselected to isolate the desired emission wavelengths (FIG. 18).Observation at 610 nm results in selective observation of the donoremission, and observation at 670 or 700 nm selects the acceptoremission.

[0113] FIGS. 19-21 show the frequency-domain data for three D-A pairs.In these data Ru-BP is always the donor. The acceptor is NB, TOTO-3 orTO-PRO-3, respectively. In the absence of acceptors, the mean Ru-BDlifetime is near 100 ns (Table IV). The Ru-BD lifetime is onlymoderately decreased by the acceptor. For instance, for any of theacceptors, a ratio of 0.03 acceptors per base pair results in a meandonor lifetime is near 70 ns. This was initially surprising given the2-fold or larger quenching of the Ru-BD intensity by these acceptorconcentrations. However, this difference in intensity and lifetimequenching can be explained as due to a range of D-to-A distances in thelabeled DNA. More specifically, most of the acceptor emission resultsfrom the more closely spaced D-A pairs. In contrast, the observed donoremission in the presence of acceptors is increased by the higherintensities of those donors most distant from acceptors, which are alsothe donors with the longer lifetimes.

[0114] The lower panels of FIGS. 19-21 shows the frequency responseobserved for the longer wavelength regions dominated by the acceptoremission. In each case the mean decay times are near 30 ns forobservation at the acceptor emission wavelengths. While the frequencyresponses are multi-exponential, visually obvious contributions from thedirectly excited acceptors with their 0.3 to 2.3 ns lifetimes were notfound. The apparent acceptor lifetimes are shorter than the apparentdonor lifetimes because the acceptor emission is enriched for theshorter distances D-A pairs which have a shorter donor lifetime.

[0115] It is informative to examine the intensity decays in thetime-domain reconstructed from the frequency-domain data (FIG. 22). Thedecays of the directly excited acceptors are short, and emission fromthe directly excited acceptors will not be observed if the detection isoff-gated for the first 10-20 ns following the excitation pulse. Thedonor decays, even in the presence of acceptors, are long lived. Also,following a brief transition period out to 10-40 ns, the acceptor decayrates are comparable to that of the quenched donors. This long livedemission from the donors can be used for biophysical or analyticalpurposes.

[0116] An important conclusion from these experiments is that theapparent acceptor decays are adequately long for off-gating of theautofluorescence from biological samples. Hence the use of MLC-acceptorpairs provides an opportunity to obtain luminophores which display longlifetimes, high quantum yields, and long emission wavelengths.

[0117] By consideration of the well known characteristics of Förstertransfer, one can predict that suitable designed D-A pairs will displayeven more favorable properties. For instance, the acceptor decay timesfor the DNA bound probes were shorter than the donor decay times. Thiseffect is due to a range of donor-to-acceptor distances for the probesrandomly bound to DNA. It is well known that unique D-to-A distances canbe obtained with polyproline spacers [53] or with double-stranded DNA asthe spacer [54-55]. In such cases the donor decay times will decrease inproportion to the transfer efficiency, and the acceptor decay times willbe similar to the donor decay times. The results for a donor andacceptor separated by a single distance are expected to be comparable tothat shown in FIG. 11, where a 1 μs decay time donor, with 90% transferefficiency, results in a luminophore with a 100 ns lifetime. Sincemetal-ligand complexes are known with decay times as long as 42 μs[56-57], one can predict 4 μs decay time luminophores with 90% transfer.

[0118] Another advantage of these RET probes is that the emissionspectra of red and NIR fluorophores are typically narrow on thewavelength scale, whereas the emission spectra of the MLCs are broad.Since autofluorescence from biological samples is typically broaderdistributed broadly on the wavelength scale, the concentration of theemission into a narrow spectral range by the acceptor will improvedetectability of these luminophores. TABLE III Quantum Yields (Q), DecayTimes (τ) and Molar Extinction Coefficients (ε/λ_(max)) of Fluorophoresin DNA Donor/ ε/λ_(ex) ε/λ_(max) Probe Acceptor Q^(a) τ (ns)(M⁻¹cm⁻¹/nm) (M⁻¹cm⁻¹/nm) AO Donor 0.392 5.0 23,300/470  53,000/500 EBDonor 0.219 21.9 5,200/518  5,200/518 RuBD Donor 0.008 84.0 13,000/440 13,000/440 NB Acceptor 0.004 0.32 1,180/440 42,900/656 TOTO-3 Acceptor0.06 2.3 2,240/440 154,000/642  TO-PRO-3 Acceptor 0.11 1.8   200/440102,000/642 

[0119] TABLE IV Multi-exponential intensity decay anlayses of the Ru-BDdonor and acceptors bound to calf thymus DNA. Donor/Acceptor n^(a)<τ>^(b) α_(i) f_(i) τ_(i) χ_(R) ² Ru-BD/NB^(c) Ru-BD 2 84 0.36 0.13 240.91 0.64 0.87 93 NB 1 0.32 1.00 1.00 0.32 0.85 DA Obs. 610 nm 2 75 0.500.15 16 1.40 0.50 0.85 86 DA Obs. 700 nm. 3 30 0.95 0.51 1.9 0.90 0.040.21 22 0.01 0.28 87 Ru-BD/TOTO-3^(c) Ru-BD 2 98 0.42 0.18 33 0.99 0.580.82 111 TOTO-3 1 2.3 1.00 1.00 2.3 1.30 DA Obs. 610 nm 2 73 0.79 0.195.8 1.07 0.21 0.81 90 DA Obs. 670 nm. 3 39 0.83 0.41 5.6 1.02 0.12 0.2423 0.05 0.35 88 Ru-BD/TO-PRO-3^(c) Ru-BD 2 114 0.42 0.17 38 1.02 0.580.83 130 TO-PRO-3 1 1.8 1.00 1.00 1.8 1.02 DA Obs. 610 nm 2 83 0.62 0.1914 0.81 0.38 0.81 99 DA Obs. 670 nm. 3 24 0.83 0.48 5.1 1.05 0.15 0.3319 0.02 0.19 78

[0120] The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples. Also, the preceding specific embodiments are to be construedas merely illustrative, and not limitative of the remainder of thedisclosure in any way whatsoever.

[0121] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

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In the claims:
 1. A luminophore comprising a donor portion (D) in closeassociation with an acceptor portion (A) sufficient for resonant energytransfer from D to A, wherein upon excitation by externalelectromagnetic radiation of a wavelength shorter than λ₁, saidluminophore emits luminophore radiation of a wavelength longer than λ₁,which is in the range of about 400 to about 1200 nm with an emissionlifetime τ₁ and a quantum yield Q₁, wherein when D is not in said closeassociation with A, it absorbs radiation of a wavelength λ₂ shorter thanλ₁ and thereafter emits radiation with a quantum yield Q₂ less thanabout 0.2, wherein when said donor portion D is in said closeassociation with A and is excited by electromagnetic radiation ofwavelength shorter than λ₁, it resonantly transfers energy to saidacceptor portion A which then resonantly emits said luminophoreradiation, and wherein said quantum yield Q₁ is substantially greaterthan Q₂.
 2. A luminophore of claim 1, which is a chemical compoundwherein D is covalently linked to A.
 3. A luminophore of claim 1,wherein each of D and A are bound to separate molecules which caninteract in solution to form said close association.
 4. A luminophore ofclaim 1, wherein said luminophore radiation has a wavelength of 550 to1000 nm.
 5. A luminophore of claim 4, wherein the emission lifetime τ₁is 25 ns to 100 μs.
 6. A luminophore of claim 5, wherein saidluminophore emission has a quantum yield Q₁ of about
 1. 7. A luminophoreof claim 6, wherein at least one of D and A comprises a functional groupby which it can be covalently bonded to another compound.
 8. A compoundof the formula D-L-Awherein D is a donor metal ligand complex having aquantum yield less than about 0.2 for emissions in the wavelength rangegreater than about 400 nm; A is an acceptor of energy resonantlytransferred from D which is then emitted in the wavelength range ofabout 400 to about 1200 nm; and L is a spacer of a length effective forresonant energy transfer between D and A.
 9. A compound of claim 2,further comprising a functional group by which it can be covalentlybonded to another compound.
 10. In a chemical compound marked with acovalently bonded detectable label, the improvement wherein the label isa compound of claim
 9. 11. A method of labeling a chemical compoundcomprising covalently bonding thereto a compound of claim
 9. 12. In amethod of identifying a chemical species in a mixture of compoundscomprising detecting radiation emitted by said chemical species, theimprovement wherein said chemical species is a compound of claim
 10. 13.A method of providing a probe which emits luminophore radiation of awavelength λ₁ in the range of about 400 nm to about 1200 nm with a highquantum yield Q₁ and a half-life greater than about 25 ns, comprisingplacing a donor molecule D, which per se emits radiation of a wavelengthless than λ₁ with a quantum yield substantially lower than Q₁, in closeassociation with an acceptor molecule A sufficient for resonant energytransfer from D to A, as a result of which D resonantly transfers energyto A and A emits said luminophore radiation.
 14. A compound of claim 8,wherein D is a transition metal ligand complex.
 15. A compound of claim14, wherein said transition metal is Re, Ru, Os or Ir.
 16. A luminophoreof claim 1, wherein D is a transition metal ligand complex.
 17. Aluninophore of claim 1, wherein said quantum yield Q₂ is about 0.1.